JP2010501178A - Shrimp pathogen diagnostic sequence - Google Patents

Abstract

  Diagnostic isolated primer for detecting infectious subcutaneous hematopoietic necrosis virus (IHHNV). This primer is based on a novel part of the IHHNV genome and can be used in primer-specific amplification or nucleic acid hybridization assays.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority from US Provisional Patent Application No. 60 / 8,865, filed Aug. 24, 2006, under 35 USC 119.

  The present invention relates to the field of diagnostic tests. More specifically, novel primers have been developed for use in detecting shrimp infectious subcutaneous hematopoietic necrosis virus pathogens.

  Commercial shrimp farms have suffered significant losses due to the effects of many common pathogens. Infectious subcutaneous hematopoietic necrosis virus (IHHNV) is one of the most serious viral pathogens of cultured prawns. This virus is widely distributed in many countries and has a broad host range. For example, IHHNV causes mass death in Western blue shrimp (Penaeus styllostris) and severe malformation in Pacific white shrimp (Penaeus vannamei). IHHNV is a parvovirus containing small single-stranded DNA, and its complete genome has been sequenced (Non-Patent Document 1).

  IHHNV causes devastating epidemics on a global scale and is extremely detrimental to the shrimp farming industry. By detecting IHHNV in hatchery seeds and late larvae, it becomes possible to remove the infected shrimp before entering the commercial production system. As a result, various methods for detecting IHHNV in shrimp have been developed, including nucleic acid-based methods and immunological methods (Non-Patent Document 2). Nucleic acid amplification methods such as polymerase chain reaction (PCR) and isothermal amplification are of particular interest because they are simple, fast and sensitive. PCR methods based on amplification of various diagnostic regions of the genome for detecting IHHNV have been described (eg, Non-Patent Document 3; Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6; and (Refer nonpatent literature 7). In addition, a loop-mediated isothermal method for detecting IHHNV has been described by Sun et al. (See Non-Patent Document 8).

Bonami et al. Gen. Virology 71 (Part 11): 657-2664 (1990); GenBank AF218266 Lightner et al., Aquaculture 164 (1): 201-220 (1998). Nunan et al., Mar. Biotechnol. 2 (4): 319-328 (2000) Yue et al. AOAC International 89 (1): 240-244 (2006) Tang et al., Dis. Aquat. Org. 44 (2): 79-85 (2001) Hu et al., CN 1410549 Dhar et al. Clin. Microbiol. 39 (8): 2835-2845 (2001) J. et al. Virol. Methods 131 (1): 41-46 (2006)

  All of the above methods are useful for detecting IHHNV, but generally lack of specificity, lack of sensitivity, or are complex and time consuming. In addition, because viruses have a high gene mutation rate, tests on different regions of the genome can be useful. Therefore, there is a need for a sensitive assay for IHHNV that is fast, accurate, and easy to use in the field. Herein, the problems pointed out are addressed by the discovery of primers based on a novel part of the IHHNV genome. By using the primers identified herein in primer-specific amplification or nucleic acid hybridization assays, IHHNV can be detected without the problems associated with prior methodologies.

  In one embodiment, the present invention provides an isolated IHHNV diagnostic primer sequence as set forth in any one of SEQ ID NOs: 1-8, or an isolated nucleic acid molecule that is completely complementary to SEQ ID NOs: 1-8. .

  In another embodiment, the present invention provides a pair of two different IHHNV diagnostic primer sequences as disclosed herein that prime a nucleic acid amplification reaction that amplifies a nucleic acid region within the IHHNV genome. Have the ability to

  In another embodiment, the present invention provides a kit for the detection of IHHNV, which comprises at least one pair of IHHNV diagnostic primer sequences disclosed herein.

In another embodiment, the present invention provides a method for detecting the presence of IHHNV in a sample, which comprises
(I) providing DNA from a sample suspected of containing IHHNV;
(Ii) probing DNA using a probe derived from any isolated IHHNV diagnostic primer sequence of SEQ ID NOs: 1-8 under suitable hybridization conditions;
Where a hybridizable nucleic acid fragment is identified that confirms the presence of IHHNV.

  In other embodiments, the detection method identifies DNA samples that are not infected with IHHNV.

In another embodiment, the present invention provides a method for detecting the presence of IHHNV in a sample, which comprises
(I) providing DNA from a sample suspected of containing IHHNV;
(Ii) amplifying the DNA with at least one pair of IHHNV diagnostic primer sequences disclosed herein so that an amplification product is generated;
Where the amplification product is present, it confirms the presence of IHHNV.

In another embodiment, the present invention provides a method for quantifying the amount of IHHNV in a sample, which comprises
(I) providing DNA from a sample suspected of containing IHHNV;
(Ii) DNA by at least one pair of IHHNV diagnostic primer sequences disclosed herein by thermal cycling at least between the denaturation temperature and the extension temperature in the presence of a nucleic acid binding fluorescent agent or fluorescently labeled probe. Amplifying the step;
(Iii) measuring the amount of fluorescence generated by the nucleic acid binding fluorescent agent or fluorescently labeled probe during thermal cycling;
(Iv) determining a threshold cycle number when the amount of fluorescence generated by the nucleic acid binding fluorescent agent or the fluorescently labeled probe reaches a fixed threshold value exceeding a basic value;
(V) By comparing the threshold cycle number determined for IHHNV in the sample with a standard curve of threshold cycle number versus logarithm of template concentration determined using a standard solution of known concentration, Calculating a quantity;
including.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS Various embodiments of the invention can be more fully understood from the following detailed description and the accompanying sequence descriptions, which form a part of this application.

A shows melting curves for the IHHNV4 product and the actin sample internal control product formed by simultaneous PCR amplification of IHHNV viral DNA and actin DNA as described in Example 10. The melting temperature (Tm) values for IHHNV and actin products are shown on their corresponding melting curves. B shows the results of separation by agarose gel electrophoresis of a sample containing the IHHNV4 product formed by simultaneous PCR amplification of IHHNV viral DNA and actin DNA and an actin sample internal control product as described in Example 10. The amounts of IHHNV and shrimp DNA are shown at the top of each lane. “M” is a 100 bp DNA ladder.

  The following sequences are subject to US Patent Law Enforcement Rules Sections 1.821-1.825 ("Requirements for Patent Applications Containing Nucleotide Sequences and / or Amino Acid Sequences-Sequence Rules Containing Nucleotide Sequences and / or Amino Acid Sequence Disclosures-the Sequence Rules)) and the World Intellectual Property Organization (WIPO) standard ST. 25 (1998) and the EPO and PCT sequence listing requirements (Rules 5.2 and 49.5 (a-2), and Article 208 of the Bylaws and Appendix C). The symbols and formats used for nucleotide and amino acid sequence data follow the rules set forth in 37 CFR 1.822.

  SEQ ID NOs: 1-8 are nucleotide sequences of IHHNV diagnostic primers useful for detecting IHHNV.

  SEQ ID NOs: 9-12 are the nucleotide sequences of the IHHNV synthesis templates described in the general methods section of the examples. These sequences are also the nucleotide sequences of amplification products obtained using the IHHNV diagnostic primer pairs disclosed herein.

  SEQ ID NOs: 13-16 are the nucleotide sequences of the sample internal control primers described in Example 10.

  SEQ ID NOs: 17 and 18 are the nucleotide sequences of fluorescently labeled probes.

  Disclosed herein are primers useful in assays for detecting infectious subcutaneous hematopoietic necrosis virus (IHHNV). By using this primer in nucleic acid amplification methods as well as hybridization assays, toxic infectious subcutaneous hematopoietic necrosis virus can be efficiently detected and quantified.

  In this disclosure, a number of terms and abbreviations are used. The definitions are provided below and are referred to for interpreting the claims and the specification.

  “Polymerase chain reaction” is abbreviated PCR.

  “Infectious subcutaneous hematopoietic necrosis virus” is abbreviated IHHNV.

  The term “isolated IHHNV diagnostic primer sequence” refers to the sequence corresponding to the portion of the IHHNV genome that is diagnosed for the presence of IHHNV.

  As used herein, an “isolated nucleic acid fragment” is a polymer of single-stranded or double-stranded RNA or DNA, optionally containing synthetic, non-natural or modified nucleotide bases. Including. An isolated nucleic acid fragment in the form of a polymer of DNA can consist of one or more segments of cDNA, genomic DNA or synthetic DNA.

  The term “amplification product” or “amplicon” refers to a nucleic acid fragment produced in a primer-specific amplification reaction. Typical primer-specific amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), or other isothermal amplification processes. When a PCR method is selected, the composition of replication typically can include, for example, deoxynucleotide triphosphate, two primers of appropriate sequence, a thermostable DNA polymerase, and a protein. Details describing these reagents and procedures for their use in nucleic acid amplification can be found in US Pat. No. 4,683,202 (1987, Mullis et al.) And US Pat. No. 4,683,195 ( 1986, Mullis et al.). When the LCR method is selected, the composition of nucleic acid replication can be determined by, for example, thermostable ligase (eg, T. aquaticus ligase), two sets of adjacent oligonucleotides (where one member of each set is Complementary to each of the target strands), may comprise Tris-HCl buffer, KCl, EDTA, NAD, dithiothreitol and salmon sperm DNA (eg, Tabor et al., Proc. Natl. Acad. Sci. U. S. A., 82: 1074-1078 (1985)).

  The term “primer” refers to an oligonucleotide (synthetic or synthetic) that has the ability to act as a starting point for nucleic acid synthesis or replication along a complementary strand when subjected to conditions in which the synthesis of the complementary strand is catalyzed by a polymerase. Natural).

  The term “thermal cycling” refers to the overall change pattern of temperature used in certain nucleic acid amplification methods such as PCR and LCR. This process is common and well known in the art. For example, Sambrook, J. et al. Fritsch, E .; F. And Maniatis, T .; , "Molecular Cloning: A Laboratory Manual", 2nd Edition, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY (1989); and U.S. Pat. No. 4,683,202 to Mullis et al. See U.S. Pat. No. 4,683,195. In general, PCR thermocycling includes an initial denaturation step at high temperature followed by a temperature cycle designed to allow template denaturation, primer annealing, and extension of the annealed primer by a polymerase.

  The term “threshold cycle number”, also referred to herein as “CT”, refers to the number of cycles in thermal cycling when the amount of fluorescence due to product formation reaches a fixed threshold above the baseline value.

  The term “probe” refers to an oligonucleotide (synthetic or natural) that is highly complementary to a target sequence, also referred to herein as a “fragment” (ie, a sequence to be detected or a portion of a sequence to be detected), and a target A duplex structure is formed by hybridizing with at least one strand of the sequence. Probes can be easily detected by labeling, for example, using fluorescent labels or ligand labels.

  The term “replication-inhibiting moiety” refers to any atom, molecule or chemical group attached to the 3′-terminal hydroxyl group of an oligonucleotide and prevents the initiation of chain extension to replicate the nucleic acid strand. Examples include, but are not limited to, 3 ′ deoxynucleotides (eg, cordycepin), dideoxynucleotides, phosphates, ligands (eg, biotin and dinitrophenol), reporter molecules (eg, fluorescein and rhodamine), carbon chains (eg, , Propanol), mismatched nucleotides or polynucleotides, or peptide nucleic acid units.

  The term “non-participating” refers to the lack of involvement of a probe or primer in a reaction to amplify a nucleic acid molecule. Specifically, non-participating probes or primers are those that do not act as a substrate for DNA polymerase or are not extended by DNA polymerase. “Non-participating probes” are essentially incapable of polymerase chain extension. This may or may not have a replication inhibiting moiety.

  A nucleic acid molecule “hybridizes” with another nucleic acid molecule, such as cDNA, genomic DNA, or RNA, when a nucleic acid molecule in single-stranded form can anneal to another nucleic acid molecule under conditions where the temperature and ionic strength of the solution are favorable. Soybean is possible. Hybridization and washing conditions are well known and are described in Sambrook, J. et al. Fritsch, E .; F. And Maniatis, T .; "Molecular Cloning: A Laboratory Manual", 2nd edition, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY (1989), especially illustrated in Chapter 11 and Table 11.1 in this document (total) As incorporated herein by reference). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. For prescreening for homologous nucleic acids, low stringency hybridization conditions corresponding to a melting temperature (Tm) of 55 ° C. can be used, for example, 5 × SSC, 0.1% SDS, 0.25% milk. And no formamide; or 30% formamide, 5 × SSC, 0.5% SDS. Moderate stringency hybridization conditions correspond to higher Tm, for example 40% formamide, 5 × or 6 × SSC. Hybridization requires that two nucleic acids contain complementary sequences, but mismatches between bases are possible depending on the stringency of the hybridization. The appropriate stringency for nucleic acid hybridization depends on variables well known in the art, such as the length of the nucleic acids and the degree of complementation. The greater the degree of similarity or homology between two nucleotide sequences, the higher the Tm value for hybrids of nucleic acids having those sequences. The relative stability of nucleic acid hybridization (corresponding to a higher Tm) decreases in the order of RNA: RNA, DNA: RNA, DNA: DNA. For hybrids longer than 100 nucleotides, a formula for Tm has been derived (see Sambrook et al., Supra, 9.50-9.51). For hybridization with shorter nucleic acids, ie oligonucleotides, the position of the mismatch becomes more important and the length of the oligonucleotide determines its specificity (see Sambrook et al., Supra, 11.7 to 11.8). . In one embodiment, the length of a hybridizable nucleic acid is at least about 10 nucleotides. Preferably, the minimum length of a hybridizable nucleic acid is at least about 15 nucleotides, more preferably at least about 20 nucleotides, and most preferably the length is at least 30 nucleotides. Furthermore, those skilled in the art will recognize that the temperature and wash solution salt concentration can be adjusted as needed according to factors such as the length of the probe.

  “Gene” refers to a nucleic acid fragment that expresses a specific protein and includes regulatory sequences before (5 ′ non-coding sequence) and after (3 ′ non-coding sequence) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Thus, a chimeric gene can comprise regulatory and coding sequences that are derived from different sources, or regulatory and coding sequences that are derived from the same source but are arranged in a manner different from that which occurs in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene that is not normally present in the host organism, but is introduced into the host organism by gene transfer. The foreign gene can comprise a natural gene inserted into a non-natural organism or a chimeric gene. A “transgene” is a gene that has been introduced into the genome by a transformation technique.

  The term “operably linked” refers to the binding of nucleic acid sequences on a single nucleic acid fragment such that one function is affected by the other. For example, a promoter is operably linked to a coding sequence if it has the ability to cause expression of the coding sequence (ie, the coding sequence is under transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

  The term “expression” as used herein refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment of the invention. Expression also refers to the translation of mRNA into a polypeptide.

  The terms “plasmid”, “vector” and “cassette” refer to extrachromosomal elements that carry genes, usually in the form of circular double-stranded DNA molecules, but are often not part of the central metabolism of the cell. Such elements may be linear or circular, single or double stranded DNA or RNA self-replicating sequences from any source, genomic integration sequences, phage or nucleotide sequences, where multiple The nucleotide sequence is linked to or recombined with a unique structure having the ability to introduce the promoter fragment and DNA sequence of the selected gene product into the cell along with the appropriate 3 ′ untranslated sequence. “Transformation cassette” refers to a specific vector comprising a foreign gene and further having elements that promote the transformation of a particular host cell in addition to the foreign gene. “Expression cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow expression of the gene in a foreign host.

  The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software includes, but is not limited to, the GCG suite program (Wisconsin Package, Version 9.0, Genetics Computer Group (GCG), Madison, WI), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol). Biol. 215: 403-410 (1990), DNASTAR (DNASTAR, Inc., Madison, Wis.), And Vector NTI® software, version 7.0. Thus, when analyzed using sequence analysis software, it will be understood that unless otherwise stated, the results of the analysis are based on the “initial values” of the referenced program. As used herein, “initial value” shall mean any set of values or parameters that are initially imported into the software when first initialized.

  Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described in Sambrook, J. et al. Fritsch, E .; F. And Maniatis, T .; "Molecular Cloning: A Laboratory Manual", 2nd Edition, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY (1989) (hereinafter "Maniatis"); and Ausubel, F. M.M. Et al., “Current Protocols in Molecular Biology”, Green Publishing Assoc. and Wiley-Interscience (1987).

Infectious Subcutaneous Hematopoietic Necrosis Virus Genome Infectious Subcutaneous Hematopoietic Necrosis Virus (IHHNV) is a major shrimp pathogen with high mortality and a broad host range. The complete genome of IHHNV has been sequenced (Bonami et al., J. Gen. Virology 71 (Part 11): 657-2664 (1990); GenBank AF218266). This genome consists of single-stranded DNA containing 4,075 bases and three open reading frames (ORFs).

IHHNV diagnostic primer sequences Disclosed herein are diagnostic primer sequences useful in various assay formats for sensitive detection of IHHNV. These primers target the region of the IHHNV genome that has not been previously used for IHHNV detection.

  Primer sequences were identified experimentally using a series of “in silica” (ie computer-based) sequence analysis tools. In this process, a database was constructed containing all known IHHNV sequences. These sequences are first aligned, then the primer sites are homologated with other IHHNV sequences using a Vector NTI® software (InforMax Inc., Bethesda, MD), specific amplicons. Analysis based on length, salt concentration, Tm (melting temperature), C + G content and absence of hairpin and secondary structure parameters. Promising primers were then screened against the GenBank sequence. Primers established to contain less than 5 bases of homology with other non-target gene sequences were selected for experimental investigation from PCR amplification efficiency and minimal primer dimer formation. A panel of DNA isolated from shrimp infected with various shrimp pathogens and shrimp-derived DNA proven to be free of disease with both high amplification efficiency and minimal primer dimer formation Selected for inspection by. Amplifies all IHHNV strains and does not react to either shrimp-derived DNA infected with non-IHHNV pathogens or DNA isolated from shrimp that has been proven not to suffer from various species of disease Primers were selected as useful primers.

  The primer sequences found to be useful for detection of IHHNV and their positions in the IHHNV genome are shown in Table 1. These primers may be synthesized using standard phosphoramidite chemistry, or purchased from companies such as Sigma Genosys (The Woodlands, TX).

Assay Methods The primer sequences disclosed herein can be used in various assay formats to detect and quantify IHHNV. The two simplest formats utilize nucleic acid hybridization methods or primer specific amplification methods such as PCR.

Primer Specific Amplification Assay In one embodiment, the present IHHNV diagnostic primer sequences can be used for primer specific nucleic acid amplification to detect the presence of IHHNV. A variety of primer-specific nucleic acid amplification methods are well known in the art and are suitable for use with the primers disclosed herein. These nucleic acid amplification methods include thermal cycling methods (eg, polymerase chain reaction (PCR) and ligase chain reaction (LCR)), and isothermal methods and strand displacement amplification (SDA).

  The LCR method is well known in the art (see, for example, Tabor et al., Proc. Natl. Acad. Sci. USA, 82: 1074-1078 (1985)). Typically, the composition of LCR nucleic acid replication consists of, for example, a thermostable ligase (eg, T. aquaticus ligase), two sets of adjacent oligonucleotide primers (one member of each set of the target strand) Complementary to each), comprising Tris-HCl buffer, KCl, EDTA, NAD, dithiothreitol and salmon sperm DNA.

  SDA methods are also well known in the art. A detailed discussion of the SDA methodology is provided by Walker et al. (Proc. Natl. Acad. Sci. USA, 89: 392 (1992)). Typically, SDA uses two oligonucleotide primers, each having a region that is complementary to only one strand in the target. After heat denaturation, the single-stranded target fragment binds to each excess primer. Both primers contain an asymmetric restriction enzyme recognition sequence located 5 'of the target binding sequence. Each primer-target complex is nicked and polymerized / polymerized in the presence of restriction enzyme, DNA polymerase and three deoxynucleotide triphosphates (dNTP) and one deoxynucleotide α-thiotriphosphate (dNTP [aS]). Repeat the replacement step.

  A preferred method for detecting IHHNV using the diagnostic primer sequences disclosed herein is PCR, which is described by Mullis et al. In US Pat. No. 4,683,202 and US Pat. No. 4,683. , 195, both of which are specifically incorporated herein by reference. In the PCR method, the IHHNV diagnostic primer sequences disclosed herein and their fully complementary sequences are used in pairs capable of priming nucleic acid amplification reactions that amplify regions within the IHHNV genome. Various combinations of IHHNV diagnostic primer sequences and their complements can be used. Suitable primer pairs include, but are not limited to, SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, and SEQ ID NOs: 1 and 4.

  In general, the two primers are mixed in a buffer solution with sample DNA, a mixture of four deoxynucleotide triphosphates (ie, dATP, dCTP, dTTP, and dGTP), a thermostable DNA polymerase, such as Taq DNA polymerase. This mixture is then subjected to thermal cycling using a thermal cycler to amplify the desired target area. Thermal cyclers are commercially available from a number of suppliers (eg, Applied Biosystems (Foster City, CA); Brinkmann (Westbury, NY); MJ Research (Waltham, Mass.); And Stratagene (La Jolla, Calif.)).

  In general, PCR thermal cycling involves an initial denaturation step at high temperature followed by a series of temperature cycles designed to allow template denaturation, primer annealing, and polymerase extension of the annealed primer. In general, the sample is first heated for about 2-10 minutes at a temperature of about 95 ° C. to denature the double-stranded DNA sample. Next, at the beginning of each cycle, the sample is denatured for about 10-60 seconds, depending on the sample and the type of instrument used. After denaturation, the primer is annealed to the target DNA at a cooler temperature of about 40 ° C. to about 60 ° C. for about 20-60 seconds. Primer extension by polymerase is often performed at a temperature range of about 60 ° C to about 72 ° C. The time required for extension may depend on the size of the amplicon and the type of enzyme used for amplification, but is easily determined by routine experimentation. In addition, the annealing step can be combined with the extension step into a two-step cycling. Thermal cycling can also include further temperature changes in the PCR assay. The number of cycles used in the assay depends on many factors, including the primers used, the amount of sample DNA present, and the thermal cycling conditions. The number of cycles used in any assay can be readily determined by one skilled in the art using routine experimentation. In some cases, a final extension step may be added after completion of thermal cycling to ensure synthesis of all amplification products.

  Following amplification, the amplified nucleotide sequence can be ligated into a suitable vector, followed by transformation of a suitable host organism with the vector. This ensures the amplified sequence in a more readily available supply form. Alternatively, after amplification, the amplified sequence or portion thereof may be chemically synthesized for use as a nucleotide probe for a hybridization assay, as described below. In any case, the DNA sequence of the variable region can be constructed using methods such as the dideoxy method (Sanger, F. et al., Proc. Natl. Acad. Sci. 74: 5463-5467 (1977)). Using the resulting sequence guides the probe options for that organism and selects the most appropriate sequence.

  In order to detect the presence of IHHNV in a sample suspected of containing IHHNV (eg, shrimp or other crustaceans) using a primer-specific nucleic acid amplification method, the DNA from the sample is provided in an amplifiable form. There must be. Typically, the DNA must be cell free and the sample material can be processed to remove proteins and other cellular components. DNA can be obtained from any suitable tissue, fluid or sample material, including but not limited to shrimp tissue (eg, heel, abdominal limb, hemolymph, muscle, tail, stalk, stomach, leg, and connective tissue ), Cleaning solution, and pond water sample. The sample can be suspected of containing IHHNV for a variety of reasons, including proximity to known contaminants, or simply because of the presence of IHHNV in the shrimp industry. Thus, the sample suspected of containing IHHNV can be any of the DNA samples described above.

  Methods for providing DNA suitable for amplification from tissues are well known in the art. For example, DNA can be homogenized with Tris-HCl buffer containing NaCl, as described by Nunan et al. (Mar. Biotechnol. 2 (4): 319-328 (2000)) The solid debris can be removed by centrifugation and the resulting supernatant can be extracted from the sample by using it in a PCR reaction. Alternatively, white spot syndrome virus DNA from various tissues described by Yoganandhan et al. (Aquaculture Research 34 (12): 1093-1097 (2003)) and Kou et al. (US Pat. No. 6,190,862). A method for isolating may be used. DNA is also amplified using commercially available DNA isolation kits such as QIAamp DNA Mini Kit (Qiagen, Valencia CA), or DNAzol® Genomic DNA Isolation Reagent (Molecular Research Center, Inc., Cincinnati, OH) May be provided in a suitable form.

  The DNA is then amplified with at least one pair of diagnostic primer sequences disclosed herein using a nucleic acid amplification method as described above. Combinations of different pairs of diagnostic primer sequences can also be used. If the presence of amplification product is detected as described below, it is a confirmation of the presence of IHHNV in the sample. In one embodiment, DNA is amplified using PCR.

  In the nucleic acid amplification method, a test result can be misinterpreted due to a defect in a reagent, a procedure error, and a malfunction of an instrument. In addition, problems arise due to the presence of inhibitors in the sample material, or degradation of sample DNA or RNA during sample processing and nucleic acid recovery. To overcome these problems, an internal control test can be performed in combination with the IHHNV assay to alert the user to this type of error and assist in quantifying the test results.

  Two types of internal control tests can be used. One approach is based on co-amplification of an “template internal control” (ITC), where ITC is added to the nucleic acid amplification reagent mixture prior to the reaction. The second approach is based on co-amplification of the “Sample Internal Control” (ISC) contained in the sample. In either case, the sequence of the internal control DNA or RNA is different from that of IHHNV DNA.

  Sample internal controls may be DNA or RNA gene sequences stored or always present in sample material (eg, shrimp tissue and hemolymph). The primers used to amplify the ISC target DNA or RNA are selected not to amplify IHHNV DNA, and the IHHNV test primer is selected not to amplify the sample internal control DNA or RNA target. In this way, the ISC target and the IHHNV target are amplified independently. In the assay, both the ISC target and the IHHNV target are processed using the same reagents and conditions. Furthermore, both target templates are amplified using the same reagents and reaction conditions. Since ISC templates and primers are present in the test sample, an ISC product should be produced during amplification. If no ISC product is formed, it indicates that the test chemistry did not function correctly and the IHHNV test result is incorrect and should not be relied upon. If correct ISC product formation occurs, it indicates that the test chemistry has worked correctly, and the processing and test reaction of the IHHNV sample appears to have worked correctly, so the IHHNV test is more accurately interpreted. Can be done.

  The ISC primer can be selected from the gene sequence of a gene encoding a structural protein, metabolic enzyme or ribosome product of a pathogen host species susceptible to IHHNV infection. For example, the ISC primer can be a gene sequence derived from a shrimp actin gene, or a shrimp 18S, 23S or 5S ribosomal gene, or other constitutive gene. Suitable examples of ISC primer pairs include, but are not limited to, SEQ ID NOS: 13, 14 and SEQ ID NOS: 15, 16 derived from the bovine shrimp (Penaeus monodon) actin 1 gene (GenBank AF100986) as shown in Table 2. Can be mentioned.

  In one embodiment, at least one pair of ISC primers is included in the nucleic acid amplification reagent mixture such that the amplification reaction produces a sample internal control product. In one embodiment, at least one pair of ISC primers is selected from the group consisting of SEQ ID NOs: 13, 14, and SEQ ID NOs: 15, 16.

  In addition, the template internal control (ITC) can be advantageously used with the IHHNV test primer to assist in the quantification of the test reaction. Primer requirements for ITC are the same as ISC primer requirements except that both ITC template and primer are added to the amplification reagent mixture. The ITC primer is selected so as not to amplify genomic DNA or RNA from a test species such as shrimp susceptible to IHHNV infection. Since the ITC template is added at a known concentration, the copy number per reaction is known. Since the ITC template is included in the amplification reagent mixture, an ITC product is produced during amplification. The amount of ITC product can vary from reaction to reaction depending on the amplification efficiency of the reaction and other variables. Since these same variables also affect IHHNV DNA amplification, the amount of IHHNV product produced will be proportional to the amount of ITC product produced in the reaction. Thus, the copy number of the IHHNV template in the assay can be inferred from the proportional relationship between the ITC initially added, the ITC product formed, and the IHHNV product produced. Relative product formation can be determined in CT units when labeled internal probes are used, or by the derivative of the melting curve at the melting temperature of each product.

  ITC primer sequences may be designed theoretically or may be derived from gene sequences from non-test species such as other viruses or genes from plants and animals that are not present in the test sample. Thus, the sample material does not contain other DNA or RNA that can be amplified by ITC primers.

  In one embodiment, at least one template internal control and at least one pair of ITC primers are included in the nucleic acid amplification reagent mixture so that the amplification reaction produces at least one ITC product.

  Various detection methods well known in the art can be used in the methods disclosed herein. These detection methods include, but are not limited to, standard native gel electrophoresis (eg, acrylamide or agarose), denaturant concentration gradient gel electrophoresis, temperature gradient gel electrophoresis, capillary electrophoresis, and fluorescence. Detection.

  The fluorescence detection method provides fast and sensitive detection of amplified products. Fluorescence detection also provides real-time detection capability where the formation of amplification products is monitored during the thermal cycling process. In addition, the amount of initial target can be quantified using fluorescence detection. Fluorescence detection can be performed by adding a nucleic acid binding fluorescent agent to the reaction mixture either before or after the thermal cycling process. Preferably, the nucleic acid binding fluorescent agent is an intercalating dye capable of non-covalent insertion between stacked base pairs in a nucleic acid duplex. However, non-intercalated nucleic acid binding fluorescent agents are also suitable. Non-limiting examples of nucleic acid binding fluorescent agents useful in the methods of the present invention are ethidium bromide and SYBR® Green I (available from Molecular Probes; Eugene, OR). By adding a nucleic acid binding fluorescent agent to the reaction mixture prior to thermal cycling, the formation of amplification products can be monitored in real time as described by Higuchi (US Pat. No. 5,994,056) It becomes. Thermal cyclers capable of real-time fluorescence measurements are commercially available from companies such as Applied Biosystems (Foster City, CA), MJ Research (Waltham, Mass.), And Stratagene (La Jolla, Calif.). Confirmation of the amplified product after amplification can be assessed by determining the melting temperature of the product using methods well known in the art, eg, by generating a melting curve using fluorescence measurements.

  Fluorescence detection of amplification products can also be achieved using other methods well known in the art, such as the use of fluorescently labeled probes. The probe comprises a sequence that is complementary to at least a portion of the amplification product. Non-limiting examples of such probes include TaqMan® probes (Applied Biosystems) and Molecular Beacons (Goel et al., J. Appl. Microbiol. 99 (3): 435-442 (2005)). It is done. For example, the gene sequences for constructing fluorescently labeled probes for use with the IHHNV primers disclosed herein are described in Primer Express® v2.0 (as described in detail in Example 11 below). Selection can be made by analysis of the IHHNV gene and test amplicon using commercially available analysis software such as Applied BioSystems Inc., Foster City, CA. The probe sequence is selected to be within the proximal end of the specific IHHNV test amplicon. Suitable probe sequences include, but are not limited to, the sequences shown in SEQ ID NOs: 17 and 18. The probe may be fluorescently labeled using methods well known in the art, such as the methods for labeling hybridization probes described below. For real-time fluorescence detection, the probe can be double labeled. For example, the 5 'end of the probe can be labeled with a fluorophore such as 6FAM ™ (Applied BioSystems) and the 3' end can be labeled with a quencher dye such as 6-carboxytetramethylrhodamine (TAMRA). In the case of a minor groove binding probe, the 3 'end can be labeled with a complex of quencher dye and minor groove binder. Fluorescently labeled probes may be obtained from commercial sources such as Applied BioSystems.

  In one embodiment, the present invention provides a method for quantifying the amount of IHHNV in a sample. In this embodiment, DNA is provided from a sample suspected of containing IHHNV as described above. DNA is amplified by at least one pair of oligonucleotide primers disclosed herein by thermal cycling at least between the denaturation temperature and the extension temperature in the presence of a nucleic acid binding fluorescent agent or fluorescently labeled probe. . During thermal cycling, the amount of fluorescence generated by the nucleic acid binding fluorescent agent or fluorescently labeled probe is measured. From the fluorescence measurement, the threshold cycle number is determined when the amount of fluorescence generated by the nucleic acid binding fluorescent agent or the fluorescent labeled probe reaches a fixed threshold value exceeding the basic value. The threshold cycle number is referred to herein as the CT number or CT value. The CT number may be determined manually or automatically by the instrument. To determine the CT number, baseline fluorescence is determined for each sample during the first amplification cycle. A mathematical algorithm is then used to set what statistically significant changes in fluorescence can be required for the fluorescence signal to exceed background. The number of cycles when the fluorescence exceeds this threshold is referred to as the CT number. Typically, the more DNA present in the sample at the beginning of thermal cycling, the fewer cycles it will spend to reach the threshold. Therefore, the CT number is inversely proportional to the initial amount of IHHNV in the sample. Once the CT number is determined for the IHHNV sample, the threshold cycle number determined for IHHNV in the sample is the threshold for the logarithm of the template concentration determined using a standard solution of known concentration as is well known in the art. By comparing with a standard curve of cycle number, the amount of IHHNV initially present in the sample can be calculated.

Nucleic Acid Hybridization Methods Basic elements of nucleic acid hybridization testing for IHHNV include DNA probes, samples suspected of containing IHHNV, and specific hybridization methods. The probe of the present invention is a single-stranded nucleic acid sequence which is complementary to the nucleic acid sequence to be detected and which can be “hybridized” therewith. Typically in hybridization methods, probe lengths can vary from as little as 5 bases to several kilobases, and this will depend on the specific test being performed. Only a part of the probe molecule needs to be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence may not be perfect. Hybridization certainly occurs between incompletely complementary molecules, so that a proportion of the bases in the hybridized region do not pair with the appropriate complementary bases.

  The DNA probes disclosed herein are derived from the above IHHNV diagnostic primer sequences. As used herein, the phrase “derived from an IHHNV diagnostic primer sequence” means that the DNA probe is an IHHNV diagnostic primer sequence, an amplification product sequence obtained therefrom using a nucleic acid amplification method, an IHHNV diagnostic primer It is meant that it can be a sequence or part of an amplification product sequence, or a fully complementary sequence of any of the aforementioned sequences. The term “portion” as used above refers to any portion shorter than the complete sequence of the IHHNV diagnostic primer sequence or the amplification product obtained therefrom. Preferably, the length of the portion for use as a probe is at least about 15 bases, more preferably at least about 20 bases. Non-limiting examples of DNA probes derived from IHHNV diagnostic primer sequences include IHHNV diagnostic primer sequences shown as SEQ ID NOS: 1-8, amplification product sequences shown as SEQ ID NOS: 9, 10, 11, and 12, And fully complementary sequences of SEQ ID NOs: 1-12.

  Detection can be facilitated by labeling the probe. Methods for attaching labels to nucleic acid probes are well known in the art. For example, probes can be labeled during synthesis by incorporating labeled nucleotides. Alternatively, probe labeling can be done by nick translation or end labeling. The label may comprise a fluorophore for fluorescence detection, or a ligand, such as biotin, which is detected using an enzyme-labeled binding molecule that binds to the ligand (eg, enzyme-labeled streptavidin) after hybridization. Is done.

  To detect the presence of IHHNV in a sample suspected of containing IHHNV, such as shrimp or other crustaceans, DNA is provided from the sample as described above. The sample DNA can be contacted with the probe before any hybridization between the probe and the target molecule occurs. Thus, the DNA is cell free and must be placed under appropriate conditions before hybridization occurs. In addition, in certain embodiments, it may be desirable to purify the DNA to remove proteins, lipids, and other cellular components. Various nucleic acid purification methods such as phenol-chloroform extraction are well known to those skilled in the art (Maniatis, supra). In addition, kits for DNA extraction and purification are available from commercial sources (eg, IsoQuick® Nucleic Acid Extraction Kit (MicroProbe Corp., Bothell, WA); and QIAamp DNA Mini Kit (Qiagen, Valencia CA). )). Prehybridization purification is particularly useful for standard filter hybridization assays.

  In one embodiment, the hybridization assay can be performed directly on the cell lysate without the need for nucleic acid extraction. This reduces the number of steps from the sample processing process and increases the speed of the assay. In order to perform such an assay on the crude cell lysate, typically a chaotropic agent is added to the cell lysate prepared as described above. Chaotropic agents stabilize nucleic acids by inhibiting nuclease activity. In addition, chaotropic agents allow sensitive and stringent hybridization of short oligonucleotide probes with DNA at room temperature (Van Ness and Chen, Nucl. Acids Res. 19: 5143-5151 (1991)). ). Suitable chaotropic agents include guanidine chloride, guanidine thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide, and cesium trifluoroacetate, among others. Typically, the chaotropic agent is present at a final concentration of about 3M. If desired, formamide can be added to the hybridization mixture, typically 30-50% by volume.

  Hybridization methods have been established and include solution (ie, homogeneous) and solid phase (ie, heterogeneous) hybridization methods. Typically, sample DNA is probed with a probe derived from the IHHNV diagnostic primer sequences disclosed herein (ie, contacted with the probe under conditions that allow nucleic acid hybridization). This involves contacting the probe and sample DNA under the appropriate concentration and temperature conditions in the presence of an inorganic or organic salt. The probe and sample nucleic acid must be contacted for a sufficiently long time so that any possible hybridization between the probe and sample nucleic acid can occur. The concentration of probe or target in the mixture can determine the time required for hybridization to occur. The higher the probe or target concentration, the shorter the incubation time required for hybridization.

  A variety of hybridization solutions can be used. Typically, these may comprise about 20-60% by volume, preferably 30% polar organic solvent. Typical hybridization solutions are about 30-50% by volume formamide, about 0.15-1 M sodium chloride, about 0.05-0.1 M buffer, eg sodium citrate, Tris HCl, PIPES or HEPES. (PH range is about 6-9), about 0.05-0.2% surfactant such as sodium dodecyl sulfate (SDS), 0.5-20 mM EDTA, FICOLL (Amersham Bioscience Inc., Piscataway, NJ ) (Molecular weight about 300-500 kilodaltons), polyvinylpyrrolidone (molecular weight about 250-500 kilodaltons), and serum albumin. Also, typical hybridization solutions include about 0.1-5 mg / mL unlabeled carrier nucleic acid, fragmented nuclear DNA (eg, calf thymus or salmon sperm DNA, or yeast RNA), and optionally About 0.5-2% by weight glycine per unit volume may also be included. Other additions such as volume exclusion agents including various polar water solvents or swelling agents (eg, polyethylene glycol), anionic polymers (eg, polyacrylate or polymethyl acrylate), and anionic sugar polymers (eg, dextran sulfate) Agents may be included.

  Nucleic acid hybridization is adaptable to various assay formats. One of the most preferred is the sandwich assay format. Sandwich assays are particularly adaptable to hybridization under non-denaturing conditions. The main element of the sandwich type assay is a solid support. The solid support is unlabeled and is absorbed or covalently bound to an immobilized nucleic acid capture probe that is complementary to a portion of the sample DNA sequence. Particularly useful probes in the present invention are those derived from the present IHHNV diagnostic sequences as described above. The captured DNA is detected using a second probe that is labeled as described above and that is complementary to a different portion of the sample DNA sequence. The label can be detected using methods well known in the art, such as fluorescence, chemiluminescence and binding pair enzyme assays.

  Hybridization methods may also be used in combination with nucleic acid amplification methods such as PCR. For example, the IHHNV diagnostic sequence can be used as a detection probe with the 3 'end blocked in either homogeneous or heterogeneous assay formats. For example, a probe generated from this sequence may be blocked at the 3 'end or non-participating and will not be extended or involved in a nucleic acid amplification reaction. In addition, the probe incorporates a label that can serve as a binding point to immobilize the probe / analyte hybrid or as a reactive ligand that acts as a reporter to generate a detectable signal. Thus, genomic DNA isolated from a sample suspected of carrying IHHNV is amplified by a standard primer-specific amplification protocol in the presence of an excess of a 3′-end blocked detection probe, and the amplified product is Produce. The probe is blocked at the 3 'end so it does not participate in or interfere with target amplification. After the last amplification cycle, the detection probe is annealed to the relevant part of the amplified DNA, and then the annealed complex is captured on the support via the reactive ligand.

  The probe is versatile and can be designed in several alternative forms. The 3 'end of the probe can be blocked from participating in the primer extension reaction by binding of a replication inhibiting moiety. Exemplary replication-inhibiting moieties include, but are not limited to, dideoxynucleotides, 3 ′ deoxynucleotides, mismatched nucleosides or nucleotide sequences, 3 ′ phosphate groups and chemical agents such as biotin, dinitrophenol, fluorescein, Examples include rhodamine and carbon chains. Replication inhibitors are covalently linked to the 3 'hydroxy group of the 3' terminal nucleotide of non-participating probes during chemical synthesis using standard cyanoethyl phosphoramidite chemistry. This process uses solid phase synthesis chemistry, where the 3 'end is covalently bound to an insoluble carrier (controlled pore glass, or "CPG"), while the newly synthesized strand is the 5' end. To grow. Within the context of the present invention, 3-deoxyribonucleotide is a preferred replication inhibitor. Cordycepin (3-deoxyadenosine) is most preferred. Since cordycepin binds to the 3 'end of the probe, synthesis is performed by cordycepin, 5-dimethoxytrityl-N-benzoyl-3-deoxyadenosine (cordycepin), 2-succinoyl-long chain alkylamino CPG covalently linked to CPG. (Glen Research, Sterling, VA). The dimethoxytrityl group is removed and the initiation of chain synthesis begins with the deprotected 5 'hydroxyl group of solid phase cordycepin. After the synthesis is complete, the oligonucleotide probe is cleaved off the solid support, leaving the free 2 'hydroxyl group of cordycepin attached to the 3' end. Other reagents can also bind to the 3 'end during the synthesis of non-participating probes and serve as replication inhibitors. These include, but are not limited to, other 3-deoxyribonucleotides, biotin, dinitrophenol, fluorescein, and digoxigenin. CPG carriers derivatized with each of these reagents are available from commercial sources (eg, Glen Research, Sterling, VA; and CLONTECH Laboratories, Palo Alto, CA).

  Alternatively, asymmetric amplification may be used to generate a strand complementary to the detection probe. Asymmetric PCR conditions for producing single-stranded DNA are similar to those described above for PCR, but the primer concentration is adjusted so that one primer is in excess and the other primer is limited. It is contemplated that this procedure will increase the sensitivity of the method. This increase in sensitivity can occur by increasing the number of single strands available for binding to the detection probe.

Assessment of risk of infection and DNA damage or IHHNV inactivation Methods disclosed for detecting and quantifying the presence of IHHNV in a sample disclosed herein assess the extent of DNA damage or IHHNV inactivation Can be used. For example, the methods disclosed herein can be used in combination with chemical treatment to improve shrimp health and development. Specifically, during the production and development of shrimp, a sample taken from a production facility, or a sample taken from a shrimp living environment, is sampled and the method disclosed herein for the presence of IHHNV. Can be used to test. If IHHNV is found, the facility and / or shrimp can be treated to kill or control the virus. Because this test is sensitive, IHHNV can be detected early before the production group is destroyed and suffers losses. Therefore, production efficiency and yield can be improved by using the methods disclosed herein in combination with chemical intervention. Examples of chemical treatment include, but are not limited to, Virkon® S fungicide (registered trademark of EI Du Pont de Nemours and Co.), peracetic acid, hydrogen peroxide, permanganate Oxidative bactericides such as potassium monopersulfate, hypochlorous acid, hypochlorite and iodine; probiotics, immunostimulants, feed supplements, and recombinant proteins / nucleic acids that interfere with viral host binding It is done. After chemical treatment, the shrimp can be sampled and retested to determine if the treatment was successful and the virus was eradicated.

  In another embodiment, it is contemplated that the primers disclosed herein may be used in various combinations to confirm the integrity and extent of damage to the viral genome caused by chemical treatment. For example, an amplification product of 480 bases can be produced by using a combination of the forward primer IHHNV1F (SEQ ID NO: 1) and the reverse primer IHHNV2R (SEQ ID NO: 4). This longer product can only be formed if the viral genome is intact and undamaged. Thus, by comparing the ratio of smaller products (SEQ ID NO: 9 or SEQ ID NO: 10) to longer products formed during amplification (or absence of longer products), the viral genome produced by chemical treatment or intervention The degree of damage can be evaluated. This can help to determine the efficacy of chemical treatment or intervention. Similarly, the integrity of longer segments of the IHHNV genome may be examined by using SEQ ID NO: 1 or SEQ ID NO: 8 in combination with other primers disclosed herein.

Detection Kit In another embodiment, the present invention provides a kit for detection of IHHNV based on a nucleic acid amplification method. The kit includes at least one pair of IHHNV diagnostic primer sequences as described above. In addition, the kit further includes the following reagents: thermostable DNA polymerase, mixture of four different deoxynucleotide triphosphates, nucleic acid binding fluorescent agent, at least one pair of sample internal control primers, at least one template A probe comprising at least one pair of internal control and template internal control primers, and a sequence complementary to a portion of at least one region of nucleic acid within the IHHNV genome capable of amplification by the IHHNV diagnostic primer sequence included in the kit At least one of the above. Kit primers and other reagents may be in various forms, such as liquid, dry, or tablets, and may be present in any one or more suitable containers such as vials, test tubes, and the like.

  In another embodiment, the present invention provides a kit for the detection of IHHNV based on a sandwich assay hybridization method. The kit includes a first element for collecting a sample from shrimp or other crustaceans suspected of having IHHNV, and a buffer for dispensing and lysing the sample. The second element includes a medium in a dry form or a liquid form for hybridizing the target nucleic acid and the probe nucleic acid, and at the same time, removing unwanted and non-hybridized forms by washing. The third element includes a solid support (eg, dipstick, bead, etc.) onto which an unlabeled nucleic acid probe derived from the isolated IHHNV diagnostic primer sequence disclosed herein is immobilized. (Or conjugated to). The fourth element includes a labeled probe that is complementary to a second different region in the same DNA strand, to which the immobilized unlabeled nucleic acid probe of the third element is hybridized. Labeled probes may also be derived from the isolated IHHNV diagnostic primer sequences disclosed herein.

  The invention is further defined in the following examples. While these examples illustrate preferred embodiments of the present invention, it should be understood that they are presented by way of example only. From the above discussion and these examples, those skilled in the art can ascertain the essential features of the invention and make various changes and modifications to the invention without departing from the spirit and spirit of the invention. Can be adapted to various applications and conditions.

Abbreviations have the following meanings: “sec” means seconds, “min” means minutes, “hr” means hours, “d” means days, “μL” means Means microliter, “mL” means milliliter, “L” means liter, “μM” means micromolar, “mM” means millimolar, “nM” means nanomolar Concentration means “M” means molar concentration, “mmol” means millimole, “μmol” means micromolar, “ng” means nanogram, “fg” means femtogram. Means “μg” means microgram, “mg” means milligram, “g” means gram, “nm” means nanometer, “mU” means millimeter , “U” means unit, “rxn” means reaction "PCR" means polymerase chain reaction, "OD" means optical density, _{"OD 260"} means the optical density measured at 260nm wavelength, _{"OD 280"} is measured at 280nm wavelength "OD _280/260 " means the ratio of OD ₂₈₀ value to OD ₂₆₀ value, "rpm" means the number of revolutions per minute, and "CT" detects the increase in fluorescence in the reaction. It means the number of cycles when the threshold is exceeded, and “SPF” means that it has been proven free of specific pathogens.

General Methods Standard recombinant DNA and molecular cloning techniques used in this example are well known in the art and are described in Sambrook, J. et al. Fritsch, E .; F. And Maniatis, T .; “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N .; Y. 1989, T. J. et al. Silhavy, M.M. L. Bennan and L. W. Enquist, “Experiments with Gene Fusions”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N .; Y. 1984, Ausubel, F .; M.M. Et al., “Current Protocols in Molecular Biology”, Green Publishing Assoc. and Wiley-Interscience, NY, 1987.

  Genome sequence analysis and primer assignment are performed by InforMax Inc. (Bethesda, MD) was achieved using Vector NTI® Software Suite.

Enzymes and reagents used herein were purchased from the following vendors:
Applied Biosystems, Foster City, CA: AmpliTaq (Catalog Number N808-0160);
New England Biolabs, Beverly, MA: Deoxynucleotide solution mixture (catalog number N0447S);
Sigma Genosys, The Woodlands, TX: Oligonucleotide;
Invitrogen Life Technologies, Carlsbad, CA: 4% agarose E-gel (catalog number G6018-02);
Qiagen, Valencia, CA: proteinase K (catalog number 19131); and RNase A, DNase free (catalog number 19101).

  In addition, kits and reagents were purchased from the following vendors: SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA; Catalog No. 4309155); and QIAamp DNA Mini Kit (Qiagen, Valencia, Catalog; Number 51304).

  All shrimp DNA samples were collected from Donald V. Lightner, Department of Veterinary Science and Microbiology, University of Arizona, The University of Arizona, Tucson, AZ 85721, USA. These include shrimp (SPF) and Penaeus monodon type baculovirus (MBV), Taura syndrome virus (TSV), white spot syndrome virus (WSSV) that have been proven to be free of disease. , P.M. Included were DNA samples from infected shrimps including P. monodon's Yellowhead disease virus (YHV), infectious subcutaneous hematopoietic necrosis virus (IHHNV) and infectious myonecrosis virus (IMNV).

Templates and Primers DNA oligonucleotide sequences for synthesizing IHHNV synthesis templates are used to infect the DNA genome of infectious subcutaneous hematopoietic necrosis virus (IHNNV) (GenBank accession number AF218266; Bonami, JR et al., J. Gen. Virology 71 (Part 11): 657-2664 (1990)) and synthesized using standard phosphoramidite chemistry or purchased commercially (Sigma Genosys Company, The Woodlands, TX). The DNA concentration and copy number of the synthetic template target and sample were measured spectrophotometrically at 260 nm (OD ₂₆₀ ). The template was diluted with purified water to a specific copy number and used as a positive control and standard for assay quantification. Table 3 presents the genomic location, SEQ ID NO, and template target length. Primer sequences useful for IHHNV detection are shown as SEQ ID NOs: 1-8.

Examples 1-4
Demonstration of an IHHNV assay using a synthetic target The purpose of these examples was to demonstrate the detection of an IHHNV synthetic template using PCR amplification with primers disclosed herein.

A template standard was prepared by serially diluting an IHHNV synthetic template (above) with DNase-free water 10-fold. In general, standard template concentrations ranged from 10 ^{7 to} 0 copies / μL. 25 μL of SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, Calif .; Cat. No. 4309155) with appropriate IHHNV forward and reverse primers as shown in Table 4, 125 nM each, and 45 μL / reaction final volume A master mixture was prepared by adding enough DNase-free water to make The master mixture was stored on ice until use.

  For each reaction, 5 μL of template standard was first added to the PCR reaction well, then 45 μL of the master mix was added. The reaction was then subjected to a thermal cycle of 40 cycles using a temperature program of 95 ° C. for 15 seconds and 60 ° C. for 1 minute with an initial denaturation step of 95 ° C. for 10 minutes. Amplification was performed in a MicroAmp optical 96 well reaction plate using an ABI PRISM 7900 thermal cycler (Applied Biosystems, Foster City, CA). During each cycle, PCR product formation was detected by monitoring the increase in fluorescence resulting from the interaction of the SYBR® Green reporter dye with the DNA amplification product. After completion of PCR, a dissociation curve (melting curve) was generated over a range of 60 ° C to 95 ° C. Data was analyzed using ABI PRISM 7900 SDS software. In addition, PCR product formation was analyzed by agarose gel electrophoresis using 4% agarose Egel (Invitrogen Life Technologies, Carlsbad, CA; catalog number G6018-02) and gel manufacturer's protocol.

  The results summarized in Table 4 demonstrate that the appropriate size amplicon product was produced for each primer set when the appropriate IHHNV template was present. The minimum detection level of the template was 2-100 copies / rxn depending on the primer used. No detectable product was produced from the sample without template.

  Amplification (CT) and amplicon product formation were inversely and directly proportional to the log of the starting template concentration, respectively.

Examples 5-8
Detection and quantification of IHHNV DNA from infected shrimp tissue The purpose of these examples was to demonstrate the detection and quantification of IHHNV in infected shrimp using a PCR assay with primers disclosed herein.

In these embodiments, using the conditions described in Examples 1-4 were amplified serial dilutions of appropriate synthetic template IHHNV DNA in the range of ¹⁰ 6 to 10 ⁰ copies per reaction (above). Using the CT value determined from each of the synthetic template concentrations, a standard curve (not shown) was generated by plotting the CT value of the 95% confidence interval against the log of the initial template copy number in the standard. The slope of this curve (ie CT versus log concentration) was then used to estimate the viral genome copy in unknown samples from the respective CT values.

  Genomic DNA from shrimp infected with Hawaiian isolate of IHHNV was used. All DNA isolated from infected shrimp pods (347 ng / μL) was serially diluted with purified water and used to provide a series of samples with DNA concentrations ranging from 1 ng / μL to 1 fg / μL of total DNA. . Negative controls included a template-free water control and two DNA shrimp samples obtained by extracting DNA from two non-infected (SPF) shrimp strains (Litopenaeus vannamei and Penaeus monodon). (50 ng / rxn).

  The diluted sample was then amplified using one of the primer pairs (see Table 5) and the same amplification, master mix, thermal cycling conditions and equipment as described in Examples 1-4. Next, the CT value of each diluted DNA sample was evaluated from the PCR amplification reaction. Next, a copy of the viral genome in the sample was estimated from the CT value and slope of the standard CT against a plot of template log concentration. PCR products were also analyzed by agarose gel electrophoresis as described in Examples 1-4.

  The results are summarized in Table 5. The table shows the IHHNV copy number per reaction with a 95% confidence interval as an average of 3 replicates. The results show that all primer sets produced the correct amplicon product size from infected shrimp DNA and detected IHHNV DNA in infected shrimp samples. Detection limits ranged from about 2 copies / rxn to about 40 copies / rxn of the viral genome, depending on the primer pair used. No amplification product was detected from the water control sample or the two SPF shrimp samples. The results obtained with the negative control sample demonstrate that this assay is not reactive against non-viral DNA from two test shrimp strains.

Example 9
The purpose of these examples is that the primers disclosed herein amplify DNA from IHHNV strains from various regions of the world but not shrimp DNA or RNA infected with other shrimp pathogens To prove that.

  In this example, shrimp DNA infected with other shrimp pathogens was used. Specifically, IHHNV strains from various geographical regions (Hawaii, Philippines, Thailand, Panama, Mexico, Mozambique and Madagascar) and non-IHHNV shrimp viruses (MBV, WSSV, YHV and IMNV) were isolated from shrimp. The released DNA samples were tested using the primers and PCR methods described in Examples 5-8.

  All IHHNV strains were detected with similar detection limits as the strains described in Examples 5-8. When examining shrimp DNA samples infected with non-IHHNV, no PCR amplification was observed. Taken together, these findings indicate that the IHHNV PCR primers and methods disclosed herein are selective for IHHNV and that these primers do not react with shrimp DNA or other shrimp viruses. It has been demonstrated.

Example 10
Detection of IHHNV DNA in combination with a sample internal control using PCR The purpose of this example is to generate an IHHNV product by using the IHHNV primer disclosed herein in combination with a sample internal control (ISC) primer. In addition it was to demonstrate that ISC products can be produced. The results presented below demonstrate that ISC primers independently amplify sample DNA and do not interfere with amplification of IHHNV DNA. The presence of the ISC product provides a marker that can be used as an indicator that a sufficient amount and quality of sample DNA has been recovered for testing.

  The ISC primer was derived from the bovine shrimp (Penaeus monodon) actin 1 gene sequence (GenBank: AF100006). To facilitate selective amplification of the IHHNV amplicon, ISC primers were designed to amplify DNA fragments larger than the target IHHNV amplicon. The sequences of the ISC primer pairs are shown as SEQ ID NOs: 13, 14 and SEQ ID NOs: 15, 16 (see Table 2).

  Samples containing IHHNV and shrimp actin DNA were prepared by serially diluting genomic DNA preparations from shrimp infected with IHHNV (Penaeus monodon) 10-fold with DNase-free water. Sample DNA content ranged from 0.1 ng to 0.1 pg per reaction. Genomic shrimp DNA (10 ng) from uninfected shrimp was then added to each IHHNV sample and negative control sample without IHHNV DNA.

  15 μL / reaction of SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA; catalog number 4309155), 125 nM for each of the IHHNV4 forward and reverse primers (SEQ ID NOs: 7 and 8, respectively), and actin In combination with a sufficient amount of primer stock solution (20 μM for each of the IHHNV primers and 10 μM for each of the actin primers) to obtain a final concentration of 32 nM for each of the two forward and reverse primers (SEQ ID NOs: 13 and 14) A master PCR mixture was prepared. DNase-free water was added to make a final volume of 25 μL / reaction. The master mixture was stored on ice until use.

  For each reaction, 5 μL of sample was first added to the PCR reaction well, followed by 25 μL of the master mix. The reaction was then subjected to a thermal cycle of 40 cycles using a temperature program of 95 ° C for 15 seconds and 60 ° C for 1 minute with an initial denaturation step of 95 ° C for 5 minutes. Amplification was performed in a MicroAmp optical 96 well reaction plate using an ABI PRISM 7900 thermal cycler (Applied Biosystems, Foster City, CA).

  During each cycle, product formation was monitored by CT values determined from the increase in fluorescence resulting from the interaction of SYBR® Green reporter dye with the DNA amplification product as described above. After 40 cycles, a dissociation curve (melting curve) was generated over a range of 60 ° C to 95 ° C. Data was analyzed using ABI PRISM 7900 SDS software. In addition, PCR product formation was analyzed by agarose gel electrophoresis using 4% agarose Egel (Invitrogen Life Technologies, Carlsbad, CA; catalog number G6018-02) and gel manufacturer's protocol.

  The results obtained using the ISC primers actin F2 (SEQ ID NO: 13) and actin R2 (SEQ ID NO: 14) are shown in FIGS. 1A and 1B, which demonstrate the simultaneous amplification of both template targets. Yes. Specific IHHNV DNA produced a 116 bp product with a melting temperature of 80 ° C. Actin ISC produced a product of 239 bp (Tm = 83.8 ° C.). IHHNV products and actin internal control products were detected based on their size and melting temperature differences by both melting curve analysis (FIG. 1A) and gel electrophoresis (FIG. 1B). In the absence of IHHNV target, at various IHHNV target concentrations, ISC product formation is as a single melting temperature peak at 83.8 ° C. (as shown in FIG. 1A), and electrophoresis (as shown in FIG. 1B). ). In all samples containing IHHNV template, specific IHHNV amplicons were detected by both melting temperature (Tm = 80 ° C.) and gel electrophoresis. These results demonstrate that the actin ISC template co-amplifies with the IHHNV template and that the detection limit of PCR amplification and PCR assay (0.1 pg IHHNV DNA) is not affected by the presence of ISC.

Example 11
Real-time detection of IHHNV DNA using fluorescently labeled probes This example demonstrates that the IHHNV primers disclosed herein can be used in combination with fluorescently labeled probes for real-time detection and quantification of IHHNV.

  Gene sequences for constructing fluorescently labeled probes were obtained from Applied BioSystems Inc. Selected by analysis of the IHHNV gene and test amplicons using Primer Express® v2.0 software purchased from (Foster City, CA 94404). The probe sequence was selected to be within the proximal end of the specific IHHNV test amplicon and was 50-110 bases long depending on the size and sequence of the amplicon. The selectivity of the probe sequence was imparted to a region having a G / C content of 30 to 80%, a C content higher than the G content, and no G at the 5 'end. In general, probe sequences were selected that had a Tm of 8-10 ° C. above the Tm of each of the test primers. Probe sequences that cross-hybridize with other species were not selected for use. The probe sequences selected to meet these criteria are listed in Table 6.

  The probe sequence was double labeled for real time detection. Two different labeling approaches were used. The 5 'end of the probe was labeled with a fluorophore (6FAM ™, Applied Biosystems). The 3 ′ end is labeled with a quencher dye or, in the case of a minor groove binding (MGB) probe, the 3 ′ end is a complex of quencher dye and minor groove binder. Labeled with Labeled probes were prepared and purchased from Applied BioSystems. The data presented below was obtained using the IHHNV4PT probe (SEQ ID NO: 18). Similarly, it is also conceivable that an IHHNV4PM probe (SEQ ID NO: 17) can be used.

A template standard was prepared by serially diluting an IHHNV synthetic template (above) with DNase-free water 10-fold. In general, standard template concentrations ranged from 10 ^{7 to} 0 copies / μL. 25 μL / reaction of TaqMan® Universal Master Mix (Applied Biosystems, Foster City, CA; catalog number 4326708) to obtain a final concentration of 125 nM for each of the appropriate IHHNV forward and reverse primers as shown in Table 7. In combination with a sufficient amount of primer stock solution (20 μM for each of the IHHNV primers), an amount of probe stock solution to obtain a final concentration of 50 nM, and sufficient DNase-free water to make a final volume of 45 μL / reaction A master mixture was prepared. The master mixture was stored on ice until use.

  For each reaction, 5 μL template standard and then 45 μL master mix was added to each PCR reaction well. The reaction was then subjected to a thermal cycle of 40 cycles using a temperature program of 95 ° C. for 15 seconds and 60 ° C. for 1 minute with an initial denaturation step of 95 ° C. for 10 minutes. Amplification was performed in a MicroAmp optical 96 well reaction plate using an ABI PRISM 7900 thermal cycler (Applied Biosystems, Foster City, CA). During each cycle, PCR product formation was detected by monitoring the increase in fluorescence resulting from the fluorescently labeled probe.

  Data was analyzed using ABI SDS 2.2 software. In addition, PCR product formation was analyzed by agarose gel electrophoresis using 4% agarose Egel (Invitrogen Life Technologies, Carlsbad, CA; catalog number G6018-02) and manufacturer's protocol.

  The results summarized in Table 7 demonstrate that the appropriate size amplicon product was produced for each primer / probe set when the appropriate IHHNV template was present. The minimum detection level of template was 50 copies / rxn depending on the primer and probe used. Samples containing no template produced no detectable product.

  Amplification (CT) and amplicon product formation were inversely and directly proportional to the log of the starting template concentration, respectively.

Claims (28)

  An isolated IHHNV diagnostic primer sequence as set forth in SEQ ID NO: 1 or an isolated nucleic acid molecule that is completely complementary to SEQ ID NO: 1.   An isolated IHHNV diagnostic primer sequence as set forth in SEQ ID NO: 2 or an isolated nucleic acid molecule that is completely complementary to SEQ ID NO: 2.   An isolated IHHNV diagnostic primer sequence set forth in SEQ ID NO: 3 or an isolated nucleic acid molecule that is completely complementary to SEQ ID NO: 3.   An isolated IHHNV diagnostic primer sequence as set forth in SEQ ID NO: 4 or an isolated nucleic acid molecule that is completely complementary to SEQ ID NO: 4.   An isolated IHHNV diagnostic primer sequence set forth in SEQ ID NO: 5 or an isolated nucleic acid molecule that is completely complementary to SEQ ID NO: 5.   An isolated IHHNV diagnostic primer sequence set forth in SEQ ID NO: 6 or an isolated nucleic acid molecule that is completely complementary to SEQ ID NO: 6.   An isolated IHHNV diagnostic primer sequence as set forth in SEQ ID NO: 7 or an isolated nucleic acid molecule that is completely complementary to SEQ ID NO: 7.   An isolated IHHNV diagnostic primer sequence set forth in SEQ ID NO: 8 or an isolated nucleic acid molecule that is completely complementary to SEQ ID NO: 8.   9. A pair of two different IHHNV diagnostic primer sequences according to any one of claims 1 to 8, having the ability to prime a nucleic acid amplification reaction that amplifies a region of nucleic acid within the IHHNV genome.   10. Two different IHHNV diagnostics according to claim 9, selected from the group consisting of SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, and SEQ ID NO: 1 and 4. Pair of primer sequences.   A kit for detecting IHHNV comprising at least one pair of the primer sequences for diagnosis of IHHNV according to claim 9.   Thermostable polymerase, mixture of four different deoxynucleotide triphosphates, nucleic acid binding fluorescent molecule, at least one pair of sample internal control primers, at least one template internal control and at least one pair of template internal control primers, and IHHNV The method further comprises at least one reagent selected from the group consisting of probes comprising sequences complementary to a portion of at least one region of nucleic acid within the IHHNV genome that can be amplified by at least one pair of diagnostic primer sequences. 10. A kit for detecting IHHNV according to 10. A method for detecting the presence of IHHNV in a sample comprising:
(I) providing DNA from a sample suspected of containing IHHNV, and (ii) for the isolated IHHNV diagnostic according to any one of claims 1-8 under suitable hybridization conditions Searching for the DNA with a probe derived from a primer sequence;
And wherein a hybridizable nucleic acid fragment is identified, it confirms the presence of IHHNV.   A group wherein the probe derived from the isolated IHHNV diagnostic primer sequence comprises SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and their fully complementary sequences 14. A method for detecting the presence of IHHNV in a sample according to claim 13 selected from.   14. The method of claim 13, wherein the probe comprises a replication inhibiting moiety at the 3 'end.   16. The method of claim 15, wherein the replication inhibiting moiety is selected from the group consisting of dideoxynucleotides, 3'deoxynucleotides, mismatched nucleosides or nucleotide sequences, 3'phosphate groups and chemical agents.   17. The method of claim 16, wherein the 3 'deoxynucleotide is cordycepin. A method for detecting the presence of IHHNV in a sample comprising:
(I) providing DNA from a sample suspected of containing IHHNV; and (ii) providing said DNA by at least one pair of IHHNV diagnostic primer sequences according to claim 9 so that an amplification product is generated. Amplifying process,
Wherein the presence of an amplification product is confirmation of the presence of IHHNV.   The method for detecting the presence of IHHNV in a sample according to claim 18, wherein the amplification step of (ii) is performed using the polymerase chain reaction.   The presence of IHHNV in the sample according to claim 18, wherein the amplification step (ii) is performed in the presence of a nucleic acid binding fluorescent agent or a fluorescent labeled probe, and the presence of the amplification product is confirmed using fluorescence detection. A way to detect.   21. The method of claim 20, wherein the fluorescently labeled probe is selected from the group consisting of SEQ ID NO: 17 and SEQ ID NO: 18.   19. The method of claim 18, wherein the sample internal control product is produced by including at least one pair of sample internal control primers in the amplification step of (ii).   23. The method of claim 22, wherein at least one pair of sample internal control primers is selected from the group consisting of SEQ ID NOs: 13,14 and SEQ ID NOs: 15,16.   19. The method of claim 18, wherein at least one pair of template internal control primers and at least one template internal control are included in the amplification step of (ii) to produce a template internal control product. A method for quantifying the amount of IHHNV in a sample comprising:
(I) providing DNA from a sample suspected of containing IHHNV;
(Ii) said DNA by at least one pair of IHHNV diagnostic primer sequences according to claim 9 by thermal cycling at least between the denaturation temperature and the extension temperature in the presence of a nucleic acid binding fluorescent agent or fluorescently labeled probe. Amplifying the process,
(Iii) measuring the amount of fluorescence generated by the nucleic acid-binding fluorescent agent or the fluorescently labeled probe during the thermal cycling;
(Iv) determining a threshold cycle number when the amount of fluorescence generated by the nucleic acid-binding fluorescent agent or the fluorescent labeled probe reaches a fixed threshold value exceeding a basic value; and (v) determining the IHHNV in the sample. Calculating the amount of IHHNV in the sample by comparing the determined threshold cycle number to a standard curve of threshold cycle number versus logarithm of template concentration determined using a standard solution of known concentration;
Including the above method.   26. The method of claim 25, wherein the fluorescently labeled probe is selected from the group consisting of SEQ ID NO: 17 and SEQ ID NO: 18.   26. A method according to any one of claims 13, 18 or 25 used for the assessment of DNA damage or IHHNV inactivation.   26. A method according to any one of claims 13, 18 or 25 used in combination with chemical treatments to improve shrimp health and development.

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